The neovascular eye diseases proliferative diabetic retinopathy and wet age-related macular degeneration are major causes of blindness through the lifespan. There is thus a critical need to find novel cellular components that could be targeted to block ocular neovascularization. The protein Ref-1 is one such component, responsible for activating redox-dependent transcription factors important for angiogenesis, and overexpressed in neovascularization. Inhibition of Ref-1?s redox function with novel small molecules blocks proliferation of ocular endothelial cells in vitro and in vivo in the laser-induced choroidal neovascularization (L-CNV) model. Ref-1 inhibition reduces signaling through hypoxia and inflammation pathways, and preliminary data reveals a novel connection to Wnt signaling. The long-term goal is to elucidate the role of Ref-1 in ocular neovascularization, and develop novel therapies targeting this protein or its pathway(s). The rationale for this research is that Ref-1 is a significant mediator of angiogenesis and inflammation, a target of multiple antiangiogenic small molecules, and a regulator of key angiogenesis factors including hypoxia-inducible factor 1? and NF-?B. The objectives in this application are to determine how Ref-1 functions as a regulator of angiogenesis and to develop new agents targeting this enzyme. The overall hypothesis is that Ref-1 activity is required for ocular angiogenesis and inflammation and that reducing the activity of Ref-1 will prevent ocular angiogenesis. Guided by exciting preliminary data, the hypothesis will be tested via two specific aims: Aim 1. Determine the Ref-1-modulated signaling pathway(s) that are key to angiogenesis and inflammation. The expression of Ref-1 in neovascularization will be assessed, and the expression and function of downstream targets (including newly identified Wnt pathway components) will be analyzed after knockdown and inhibition of this protein in endothelial cells with or without overexpression of functional mutants. Angiogenic activity will also be assessed. Aim 2. Optimize the preclinical profile of Ref-1 inhibitors in vitro and in vivo. Two novel, highly potent Ref-1 small molecule inhibitors will be explored for efficacy in vitro, and in cell and in multiple in vivo models of neovascularization, including synergy with anti-vascular endothelial growth factor therapy, target engagement and off-target effects, effects on Ref-1 target genes, and toxicity. This work is innovative, as it is the first in-depth mechanistic study of the role of Ref-1 in ocular angiogenesis, exploring this unique signaling node as an integrator of proangiogenic, proinflammatory, and newly identified Wnt signals. It will also reveal new signaling pathways relevant to angiogenesis and inflammation in the eye, and novel therapeutic leads for neovascular eye diseases. The work is highly significant because it will define Ref-1 as an ocular angiogenic mediator and determine its downstream effects, leading to development of new ways to prevent blindness. Additionally, outcomes from these studies will be the advancement of novel, anti-Ref-1 small molecule inhibitors for translation from the bench to the clinic and patient care.